Desaturase antigen of mycobacterium tuberculosis

ABSTRACT

The use of genetic methodology based on the fusion of the proteins with the alcaline phosphatase (Lim et al., 1995) has allowed the isolation of a new exported protein of  M. tuberculosis . In the present article, first of all the isolation of a gene encoding this exported protein called DES is described as well as its characterization and its distribution among the different mycrobacterial species. It is notably shown that the protein has in its primary sequence amino acids only found at the level of active sites of enzymes of class II diiron-oxo proteins family. Among the proteins of this family, DES protein of  M. tuberculosis  does not present significative homologies with stearoyl ACP desaturases. Secondly, the antigenic feature of this protein has been studied. For this, DES protein of  M. tuberculosis  has been overexpressed in  E. coli  under recombinant and purified protein form from this bacterium. The reactivity of tuberculous patients sera infected by  M. tuberculosis  or  M. bovis  against DES protein in Western blot experimentations has been tested. 100% of the tested patients did recognize the protein. The intensity of the antibody response against DES protein measured by ELISA of tuberculous patients sera compared with the one relating to sera patients suffering from other pathologies show that there is a significative difference between the intensity of the antibody responses of these two categories of patients. Accordingly, DES protein is a potentially interesting tool for the tuberculosis serodiagnostic.

This is a division of application Ser. No. 09/230,485, filed Apr. 20,1999 (now U.S. Pat. No. 6,582,925), which is a § 371 of PCT/IB97/00923,filed Jul. 25, 1997, and claims the benefit of U.S. ProvisionalApplication No. 60/022,713, filed Jul. 26, 1996, the disclosures of allof which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Tuberculosis and leprosy, caused by the bacilli from the Mycobacteriumtuberculosis complex and M. leprae respectively are the two majormycobacterial diseases. Pathogenic mycobacteria have the ability tosurvive within host phagocytic cells. From the interactions between thehost and the bacteria results the pathology of the tuberculosisinfection through the damages the host immune response causes on tissues(Andersen & Brennan, 1994). Alternatively, the protection of the host isalso dependent on its interactions with mycobacteria.

Identification of the bacterial antigens involved in these interactionswith the immune system is essential for the understanding of thepathogenic mechanisms of mycobacteria and the host immunologicalresponse in relation to the evolution of the disease. It is also ofgreat importance for the improvement of the strategies for mycobacterialdisease control through vaccination and immunodiagnosis.

Through the years, various strategies have been followed for identifyingmycobacterial antigens. Biochemical tools for fractionating andanalysing bacterial proteins permitted the isolation of antigenicproteins selected on their capacity to elicit B or T cell responses(Romain et al., 1993; Sorensen et al., 1995). The recent development ofmolecular genetic methods for mycobacteria (Jacobs et al., 1991; Snapperet al., 1990; Hatful, 1993; Young et al., 1985) allowed the constructionof DNA expression libraries of both M. tuberculosis and M. leprae in theλgt11 vector and their expression in E. coli The screening of theserecombinant libraries using murine polyclonal or monoclonal antibodiesand patient sera led to the identification of numerous antigens(Braibant et al., 1994; Hermans et al., 1995; Thole & van der Zee,1990). However, most of them turned out to belong to the group of highlyconserved heat shock proteins (Thole & van der Zee, 1990; Young et al.,1990).

The observation in animal models that specific protection againsttuberculosis was conferred only by administration of live BCG vaccine,suggested that mycobacterial secreted proteins might play a major rolein inducing protective immunity. These proteins were shown to inducecell mediated immune responses and protective immunity in guinea pig ormice model of tuberculosis (Pal & Horwitz, 1992; Andersen, 1994; Haslowet al., 1995). Recently, a genetic methodology for the identification ofexported proteins based on PhoA gene fusions was adapted to mycobacteriaby Lim et al. (1995). It permitted the isolation of M. tuberculosis DNAfragments encoding exported proteins. Among them, the already known 19kDa lipoprotein (Lee et al., 1992) and the ERP protein similar to the M.leprae 28 kDa antigen (Berthet et al., 1995).

SUMMARY OF THE INVENTION

We have characterized a new M. tuberculosis exported protein named DESidentified by using the PhoA gene fusion methodology. The des gene,which seems conserved among mycobacterial species, encodes an antigenicprotein highly recognized by human sera from both tuberculosis andleprosy patients but not by sera from tuberculous cattle. The amino acidsequence of the DES protein contains two sets of motifs that arecharacteristic of the active sites of enzymes from the class IIdiiron-oxo protein family. Among this family, the DES protein presentssignificant homologies to soluble stearoyl-ACP desaturases.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention will be further clarified by the following examples, whichare intended to be purely exemplary of the invention.

Bacteria, Media and Growth Conditions

The bacterial strains and plasmids used in this study are listed in FIG.8 E. coli DH5α of BL21(DE3)pLysS cultures were routinely grown in LuriaB medium (Difco) at 37° C. Mycobacterium cultures were grown inMiddlebrook 7H9 medium (Difco) supplemented with Tween 0.05%, glycerol(0.2 %) and ADC (glucose, 0.2 %; BSA fraction V, 0.5 %; and NaCI, 0.085%) at 37° C. Antibiotics when required were added at the followingconcentrations: ampicillin (100 μg/mI), kanamycin (20 μg/ml).

Human and Cattle Sera

Serum specimens from 20 individuals with pulmonary or extra-pulmonarytuberculosis (M. tuberculosis infected) were obtained from the Blignysanatorium (France). 6 sera from M. bovis infected human tuberculouspatients and 24 sera from BCG-vaccinated patients suffering from otherpathologies were respectively obtained from Institut Pasteur,(Madagascar), and the Centre de Biologie Médicale spécialisée (CBMS)(Institut Pasteur, Pads). Sera from tuberculous cattle (M. bovisinfected) were obtained from CNEVA, (Maison Alfort).

Subcloning Procedures

Restriction enzymes and T4 DNA ligase were purchased from Gibco/BRL,Boehringer Mannheim and New England Biolabs. All enzymes were used inaccordance with the manufacturers recommendations. A 1-kb ladder of DNAmolecular mass markers was from Gibco/BRL. DNA fragments used in thecloning procedures were gel purified using the Geneclean II kit (BIO 101Inc., La Jolla, Calif.). Cosmids and plasmids were isolated by alkalinelysis (Sambrook et al., 1989). Bacterial strains were transformed byelectroporation using the Gene Pulser unit (Bio-Rad Laboratories,Richmond, Calif.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the 4.5 kb EcoRV fragment encoding the M.tuberculosis des gene.

FIG. 2 shows the nucleotide (SEQ ID NO:1) and derived amino acid (SEQ IDNO:2) sequences of the M. tuberculosis des gene.

FIG. 3 shows a comparative sequence analysis of class II diiron-oxoproteins and the M. tuberculosis Des protein. Shaded residues indicatecluster ligands and probable iron ligands in the M. tuberculosis Desprotein. Bold unshaded framed letters are probable residues involved inthe network of hydrogen bonds to the cluster. Other bold lettersindicate conserved residues that are believed to participate in theO₂-binding site. Gaps introduced into the sequence of Des are indicatedby dots. Accession numbers are as follows: ribonucleotide reductases:v01555, Epstein-barr virus; k02672, E. Coli. Methane monooxygenasehydroxylases: M58499, Methylococcus capsulatus; X55394, mmoXMethylosinus trichosporium; M60276, Pseudomonas sp. strain CF 600 phenolhydroxylase dmpN polypeptide; M65106, Pseudomonas mendocina KR1.Stearoyl-ACP desaturases: M59857, Ricinus communis; M59858, cucumber;M61109, safflower; X62898, spinach; X60978, Brassica; M91238, potato;X70962, linseed; M93115, coriander Delta-4 desaturase.

FIG. 4 is a Southern blot analysis of the distribution of the des genein other mycobacterial species. DNA from various mycobacterial strainswere Pstl-digested, electrophoresed, transferred onto a nylon membraneby Southern blotting, and hybridized using probe B, which is shown inFIG. 1.

FIG. 5 shows an SDS-PAGE gel of soluble and insoluble extracts from E.coli expressing the DES protein on plasmid pETdes (I-1718).

FIG. 6 shows the results of ELISAs of the sensitivity of the antibodyresponse to the DES antigen of human tuberculous and non-tuberculouspatients.

FIG. 7 shows the nucleotide and derived amino acid sequence of theMycoplasma tuberculosis des gene. The underlined sequences correspond tothe −35 and −10 boxes of the promoter and a Shine Dalgarno sequence thatcorresponds to the putative ribosomal attachment site, respectively. Theadenosine labelled “+1” corresponds to the transcription initiationsite.

FIG. 8 is a table of the bacterial strains and plasmids used in thisapplication.

FIG. 9 is a Western blot showing the recognition of the purified DESprotein by antibodies from M. bovis and M. tuberculosis-infected humansand cattle.

Southern Blot Analysis and Colony Hybridization

DNA fragments for radiolabeling were separated on 0.7% agarose gels(Gibco BRL) in a Tris-borate-EDTA buffer system (Sambrook et al., 1989)and isolated from the gel by using Geneclean II (BIO 101). Radiolabelingwas carried out with the random primed labeling kit Megaprime (Amersham)with 5 μCi of (α-³²P)dCTP, and nonincorporated label was removed bypassing through a Nick Column (Pharmacia). Southern blotting was carriedout in 0.4 M NaOH with nylon membranes (Hybond-N+, Amersham) accordingto the Southern technique (Southern, 1975), prehybridization andhybridization was carried out as recommended by the manufacturer usingRHB buffer (Amersham). Washing at 650° C. was as follows: two washeswith 2×SSPE (150 mM NaCI, 8.8 mM NaH₂PO₄, 1 mM EDTA pH 7.4)-SDS 0.1% of15 minutes each, one wash with 1×SSPE-SDS 0.1 % for 10 minutes, twowashes with 0.7×SSPE-SDS 0.1% of 15 minutes each. Autoradiographs wereprepared by exposure with X-ray film (Kodak X-Omat AR) at −80° C.overnight. Colony hybridization was carried out using nylon membranediscs (Hybond-N+0.45 μm, Amersham). E. coli colonies adsorbed on themembranes were lysed in a (0.5 M NaOH, 1.5 M NaCI) solution, beforebeing placed for one minute in a micro-wave oven to fix the DNA.Hybridization and washings were as described for the Southern blottinganalysis.

DNA Sequencing and Analysis

Sequences of double-stranded plasmid DNA were determined by thedideoxy-chain termination method (Sanger et al., 1977) using the Taq DyeDeoxy Terminator Cycle sequencing Kit (Applied Biosystems), on a GeneAmpPCR System 9600 (Perkin Elmer), and run on a DNA Analysis System-Model373 stretch (Applied Biosystems). The sequence was assembled andprocessed using DNA strider™ (CEA, France) and the University ofWisconsin Genetics Computer Group (UWGCG) packages. The BLAST algorithm(Altschul et al., 1990) was used to search protein data bases forsimilarity.

Expression and Purification of the DES Protein in E. coli

A 1043 bp NdeI-BamHI fragment of the des gene was amplified by PCR usingnucleotides JD8 (5′-CGGCATATGTCAGCCAAGCTGACCGACCTGCAG-3′)(SEQ ID NO: 3)and JD9 (5′-CCGGGATCCCGCGCTCGCCGCTCTGCATCGTCG-3′)(SEQ ID NO: 4), andcloned into the NdeI-BamHI sites of pET14b (Novagen) to generatepET-des. PCR amplifications were carried out in a DNA thermal Cycler(Perkin Elmer), using Taq polymerase (Cetus) according to themanufacturer's recommendations. PCR consisted of one cycle ofdenaturation (95° C., 6 mm) followed by 25 cycles of amplificationconsisting of denaturation (95° C., 1 mm), annealing (57° C., 1 mm), andprimer extension (72° C., 1 mm). In the pET-des vector, the expressionof the des gene is under control of the T7 bacteriophage promoter andthe DES antigen is expressed as a fusion protein containing sixhistidine residues. Expression of the des gene was induced by additionof 0.4 mM IPTG in the culture medium. The DES protein was purified byusing a nickel-chelate affinity resin according to the recommendationsof the supplier (Qiagen, Chatsworth, Calif.). Linked to the localizationof the DES protein in cytoplasmic inclusion bodies, the purification wascarried out under denaturating conditions in guanidine hydrochloridebuffers. The protein was eluted in buffer A (6 M guanidinehydrochloride, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8) containing 100 mM EDTA.The purified protein was kept and used in buffer A, as all attempts tosolubilize it in other buffers were unsuccessful.

SDS-PAGE and Immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wascarried out as described by Laemmli (1970). For Western blottingexperiments (immunoblotting), approximately 10 μg of DES purifiedprotein were run on a SDS-polyacrylamide gel and transferred ontonitrocellulose membranes (Hybond C extra, Amersham) using a Bio-Rad minitransblot apparatus according to the recommendations of the manufacturer(Bio-Rad Laboratories, Richmond, Calif.). Transfer yield was visualizedby transient staining with Ponceau Rouge. The membrane were incubatedwith human patient or cattle sera diluted 1/200^(θ) at 37° C. for 1 hourand with a goat anti-human (Promega) or rabbit anti-cattle (Biosys)IgGalkaline phosphatase-conjugated secondary antibody diluted 1/2500^(θ)for 30 minutes at 37° C. The color reaction was performed by addition of5-bromo-4-chloro-3-indolylphosphate (0.165 mg/ml) and toluidinumnitroblue tetrazolium (0.33 mg/ml) as substrates.

ELISA

The human or cattle sera were tested for antibodies against DES byenzyme-linked immunosorbent assay (ELISA). The 96-well micro-titer trays(Nunc) were coated with 0.1 μg (per well) of purified DES protein inguanidine hydrochloride buffer A (6 M guanidine hydrochloride, 0.1 MNaH₂PO₄, 0.01 M Tris, pH 8) (1 h at 37° C. and 16 h at 4° C.). Afterthree washes, wells were saturated with bovine serum albumin 3% inphosphate buffered saline (PBS) for 30 min at room temperature. Afterthree washes, sera diluted from 1/50^(e) to 1/3200^(e) in buffer (PBS,0.1% Tween 20, 1% bovine serum albumin) were added to the wells for 2hat 37° C. After three washes, the wells were treated with goatanti-human IgG-alkaline phosphatase conjugate (Promega) diluted1/4000^(e) for 1 h at 37° C. Then, 4 mg of p-nitrophenylphosphate per mlwere added as substrate. After 20 min of incubation at 37° C., theplates were read photometrically at an optical density of 405 min inmicro-ELISA Autoreader (Dynatech, Marnes la Coquette, France).

Statistics

Antibody response of the different sera tested were compared by usingthe Student t test. P≧0.05 was considered nonsignificant.

Nucleotide Sequence and Accession Number

The nucleotide sequences of des has been deposited in the GenomeSequence Data Base (GSDB) under the accession number U49839.

Cloning of the des Gene

The construction of a library of fusions of M. tuberculosis genomic DNAto the phoA gene and its expression in M. smegmatis, described by Lim etal. (1995), led to the isolation of several PhoA+ clones. pExp421 is theplasmid harboured by one of the PhoA+ clones selected from this library.Detection of enzymatically active alkaline phosphatase indicated thatthe pExp421 insert contains functional expression and exportationsignals. Restriction analysis showed that pExp421 carries a 1.1 kbinsert. Partial determination of its sequence identified a 577 bp ORF,named des, fused in frame to the phoA gene and presenting two motifs, of9 and 14 amino acids, conserved with soluble stearoyl-acyl-carrierprotein desaturases (Lim et al., 1995).

To isolate the full-lengh des gene, the M. tuberculosis H37Rv pYUB18genomic cosmid library (Jacobs et al., 1991), was screened by colonyhydridization with the 1.1 kb probe (probe A, see FIG. 1). Twohybridizing cosmids named C₃ and C₄ were selected for further isolationof the gene. C₃ and C₄ were cut with several restriction enzymes andsubjected to Southern blot analysis using the 1.1 kb fragment as aprobe.

The EcoRV restriction profile revealed a single hybridizing fragment of4.5 kb which was subcloned into pBluescript KS⁻ (Stratagene) to giveplasmid pBS-des.

Characterization of the des Gene

The DNA sequence of the full des ORF was determined (FIG. 2). The desgene was shown to cover a 1017 bp region, encoding a 339 amino acidprotein with a calculated molecular mass of 37 kDa. The ORF starts witha potential ATG start codon at position 549, and ends with a TAG stopcodon at position 1565. There is a potential Shine-Dalgarno motif(GGAGG) at position −8 upstream of the ATG. The G+C content of the ORF(62%) is consistent with the global GC content observed in mycobacterialgenome. The nucleotide and deduced amino acid sequences of the des genewere compared to sequences in databases. They showed very highhomologies to the M. leprae aadX gene located on cosmid B2266, depositedin GenBank as part of the M. leprae genome sequencing project (GenBankaccession number n° U15182). Within the coding region, the DNA sequenceswere 79% identical while the encoded proteins were 80% identical (88%including conserved residues). The des gene also scored significantlyagainst soluble stearoyl-ACP desaturases: 44% identity at the nucleotidelevel, 30% identity (51% including conserved residues) at the amino acidlevel, to the Oryza sativa stearoyl-ACP desaturase (accession n°D38753).

Although the detection of a phoA enzymatical activity in the M.smegmatis clone harbouring the pExp421 suggests the DES protein isexported, no structural similarities were found between the DES proteinN terminal amino acids and signal sequences of bacterial exportedproteins (Izard & Kendall, 1994).

Like in M. leprae genome, a second ORF presenting high homologies to theM. leprae putative NtrB gene (cosmid B2266), is located downstream ofthe des gene in M. tuberculosis FIG. 2. Interestingly, the two ORF, desand “NtrB”, are separated in M. tuberculosis by two direct repeats of 66nucleotides overlapping on 9 nucleotides (FIG. 2). Although M. lepraeand M. tuberculosis seem to share the same genomic organization in thispart of the chromosome, these repeats are absent from the M. lepraegenome.

The des Protein Presents the Conserved Amino Acid Motifs of the Class IIDiiron-oxo Proteins

Further analysis of the amino-acid sequence of the DES protein revealedthe presence of conserved motifs found only in class II diiron-oxoproteins (Fox et al., 1994) (FIG. 3). These proteins are oxo-bridgeddiiron clusters (Fe—O—Fe) containing proteins. They possess in theirsecondary structure 4 alpha helices involved in the protein-derivedcluster ligands. As revealed by X-ray structure studies, in theseproteins, the diiron axis is oriented parallel to the long axis of thefour helix bundle with ligands arising from four noncontiguous helices,B, C, E and F. M. tuberculosis DES protein appears to have the sameactive site residues as the class II diiron-oxo enzymes. This includesGlu and His residues (E₁₀₇ and H₁₁₀ in helix C, E₁₆₇ in helix E and E₁₉₇and H₂₀₀ in helix F) that are ligands to the iron atoms, Asp, Glu andArg residues (E₁₀₆ and R₁₀₉ in helix C, D₁₉₆ in helix F) that areinvolved in a hydrogen-bonding network to the cluster and, lie and Thrresidues that may be part of the O₂-binding site (T₁₇₀ in helix E, I₁₉₃in helix F). Thus, the M. tuberculosis DES protein contains in itsprimary sequence two conserved D/E(ENXH) motifs separated by 85 aminoacids.

The class II diiron-oxo protein family contains up to dateribonucleotide reductases, hydrocarbon hydroxylases (methanemonooxygenase, toluene-4-monooxygenase and phenol hydroxylase) andsoluble-ACP desaturases. On the overall sequence alignment the DESprotein presents higher homology to soluble stearoyl-ACP desaturasesthan to ribonucleotide reductases or bacterial hydroxylases. Thepercentage identity at the amino acid level of the DES protein was saidto be 30% with the Oryza sativa stearoyl-ACP desaturase, whereas it isonly 17% with the Methylococcus capsulatus methane monooxygenase(accession n° M58499), 17.5% with the Pseudomonas sp CF 600 phenolhydroxylase (accession n° M60276) and 17.7% with the Epstein Barrribonucleotide reductase (accession n° V01555). Homologies to thesoluble Δ9 desaturases mostly concern the amino acids located within theactive site in helices C, E and F (FIG. 3).

Distribution of the des Gene in Other Mycobacterial Species

The presence of the des gene in Pstl-digested chromosomal DNA fromvarious mycobacterial strains was analyzed by Southern blotting (FIG.4). The probe used (probe B) is a PCR amplification productcorresponding to nucleotides 572 to 1589 (see FIG. 1). The probehybridized on all mycobacterial genomic DNA tested. Strong signals weredetected in M. tuberculosis, M. bovis, M. bovis BCG, M. Africanum and M.avium. Weaker signals were visible in M. microti, M. xenopi, M.fortuitum and M. smegmatis. Thus, the des gene seems to be present insingle copy at least in the slow growing M. tuberculosis, M. bovis, M.bovis BCG, M. Africanum, M. avium and M. xenopi as well as in the fastgrowing M. smegmatis.

Expression of the des Gene in E. coli

In order to overexpress the DES protein, the des gene was subcloned intothe bacteriophage T7 promoter-based expression vector pET14b (Novagen).A PCR amplification product of the des gene (see material and methods)was cloned into the Ndel-BamHI sites of the vector, leading to plasmidpET-des. Upon IPTG induction of E. coli BL21 DE3 pLysS cells harbouringthe plasmid pET-des, a protein of about 40 kDa was overproduced. Thesize of the overproduced protein is in agreement with the molecular masscalculated from the deduced polypeptide. As shown in FIG. 5, the greatmajority of the overproduced DES protein is present in the insolublematter of E. coli cells. This probably results from the precipitation ofthe over-concentrated protein in E. coli cytoplasm thus forminginclusion bodies. To be able to dissolve the protein, the purificationwas carried out using a nickel chelate affinity resin under denaturatingconditions in guanidine hydrochloride buffers. Among all the conditionstested (pH, detergents . . . ), the only condition in which the proteincould be eluted without precipitating. in the column and remain soluble,was in a buffer containing 6 M guanidine hydrochloride.

Immunogenicity of the DES Protein After Infection

20 serum samples from M. tuberculosis infected human patients (4 withextra-pulmonary tuberculosis, 15 with pulmonary tuberculosis and 1 withboth forms if the disease), 6 sera from M. bovis infected human patientsand 4 sera from M. bovis infected cattle were tested either pooled ortaken individually in immunoblot experiments to determine the frequencyof recognition of the purified DES protein by antibodies from infectedhumans or cattle. 20 out of the 20 sera from the M. tuberculosisinfected human patients and 6 out of the 6 sera from the M. bovisinfected human patients recognized the recombinant antigen as shown bythe reaction with the 37 kDa band (FIG. 9). Furthermore, a pool of serafrom human lepromatous leprosy patients also reacted against the DESantigen.

In contrast, the pool of serum specimens from M. bovis infected cattledid not recognize the DES protein. These results indicate that the DESprotein is highly immunogenic in tuberculosis human patients. Bothpulmonary and extra-pulmonary tuberculosis patients recognize theantigen.

Magnitude of Human Patients Antibody Response

An enzyme-linked immunosorbent assay (ELISA) was used to compare thesensitivity of the different serum samples from 20 tuberculosis patients(15 infected by M. tuberculosis and 5 infected by M. bovis) to the DESantigen. This technique was also carried out to compare the sensitivityof the antibody response to DES of the 20 tuberculosis patients to theone of 24 patients (BCG-vaccinated) suffering from other pathologies. Asshown on FIG. 6, patients suffering from other pathologies thantuberculosis, react at a low level to the DES antigen (averageOD₄₀₅=0.17 for a serum dilution 1/100^(e)). The average antibodyresponse from the tuberculosis patients infected by M. tuberculosis orM. bovis against the same antigen is much more sensitive (OD₄₀₅=0.32 andOD₄₀₅=0.36 respectively, for a serum dilution 1/100^(e)). Thisdifference in the sensitivity of the immunological response isstatistically highly significant at every dilution from 1/50^(e) to1/3200^(e) as shown by a Student t₉₅ test (t₉₅=5.18, 6.57, 6.16, 5.79,4.43, 2.53 and 1.95, at sera dilutions 1/50^(e), 1/100^(e), 1/200^(e),1/400^(e), 1/800^(θ), 1/1600^(e) and 1/3200^(e), respectively).

No differences in the sensitivity of the antibody response was noticedbetween patients suffering from pulmonary or extra-pulmonarytuberculosis.

The PhoA gene fusion methodology permitted the identification of a newM. tuberculosis exported antigenic protein.

This 37 kDa protein contains conserved amino acid residues which arecharacteristic of class II diiron-oxo-proteins. Proteins from thatfamily are all enzymes that require iron for activity. They includeribonucleotide reductases, hydrocarbon hydroxylases and stearoyl-ACPdesaturases. The M. tuberculosis DES protein only presents significanthomologies to plant stearoyl-ACP desaturases (44% identity at thenucleotide level, and 30% identity at the amino-acid level) which arealso exported enzymes as they are translocated across the chloroplasticmembranes (Keegstra & Olsen, 1989). This result suggests that the DESprotein could be involved in the mycobacterial fatty acid biosynthesis.Furthermore, the localization of the protein outside the cytoplasm wouldbe consistent with its role in the lipid metabolism, since lipidsrepresent 60% of the cell wall constituents and that part of thebiosynthesis of the voluminous mycolic acids containing 60 to 90 carbonatoms occurs outside the cytoplasm. Among all the different steps of thelipid metabolism, desaturation reactions are of special interest, firstbecause they very often take place at early steps of lipid biosynthesisand secondly because, through the control they have on the unsaturationrate of membranes, they contribute to the adaptation of mycobacteria totheir environment (Wheeler & Ratledge, 1994). An enzyme system involvinga stearoyl-Coenzyme A desaturase (analog of the plantstearoyl-ACP-desaturases), catalyzing oxydative desaturation of the CoAderivatives of stearic and palmitic acid to the corresponding Δ9monounsatured fatty acids has been biochemically characterized inMycobacterium phlei (Fulco & Bloch, 1962; Fulco & Bloch, 1964;Kashiwabara & al., 1975; Kashiwabara & Sato, 1973). This system wasshown to be firmly bound to a membranous structure (Fulco & Bloch,1964). Thus, M. tuberculosis stearoyl-Coenzyme A desaturase (Δ9desaturase) is expected to be an exported protein. Sonicated extracts ofE. coil expressing the DES protein were assayed for Δ9 desaturatingactivity according to the method described by Legrand and Besadoun(1991), using (stearoyl-CoA) ¹⁴C as a substrate. However, no Δ9desaturating activity could be detected. This result is probably linkedto the fact desaturation systems are multi-enzyme complexes involvingelectron transport chains and numerous cofactors, often difficult torender functional in vitro. E. coli and mycobacteria being verydifferent from a lipid metabolism point of view, the M. tuberculosisrecombinant Δ9 desaturase might not dispose in E. coil of all thecofactors and associated enzymes required for activity or might notinteract properly with them. Moreover, not all cofactors involved in theΔ9 desaturation process of mycobacteria are known, and they might bemissing in the incubation medium.

However, if the DES protein encodes a Δ9 desaturase, an amazing pointconcerns its primary sequence. Indeed, all animal, fungal and the onlytwo bacterial A9 desaturases sequenced to date (Sakamoto et al., 1994)are integral membrane proteins which have been classified into a thirdclass of diiron-oxo proteins on the basis of their primary sequencesinvolving histidine conserved residues (Shanklin et al., 1994). Theplant soluble Δ9 desaturases are the only desaturases to possess thetype of primary sequence of class II diiron-oxo proteins (Shanklin &Somerville, 1991). No bacteria have yet been found which have a planttype Δ9 desaturase.

As shown by immunoblotting and ELISA experiments, the DES protein is ahighly immunogenic antigen which elicits B cell response in 100% of thetuberculosis M. bovis or M. tuberculosis-infected human patients tested,independently of the form of the disease (extrapulmonary or pulmonary).It also elicits an antibody response in lepromatous leprosy patients.Interestingly, although more sera would need to be tested, tuberculouscattle do not seem to recognize the DES antigen. Furthermore, the ELISAexperiments showed that it is possible to distinguish tuberculosispatients from patients suffering from other pathologies on the basis ofthe sensitivity of their antibody response to the DES antigen. The DESantigen is therefore a good candidate to be used for serodiagnosis oftuberculosis in human patients. The reason why the non-tuberculouspatients tested recognize at a low level the DES protein could be due tothe fact they are all BCG-vaccinated individuals (BCG expressing theprotein), or to a cross-reactivity of their antibody response with otherbacterial antigens. It would now be interesting to know whether the DESantigen possesses, in addition to its B cell, epitopes, T cell epitopeswhich are the only protective ones in the host immunological responseagainst pathogenic mycobacteria. If the DES protein is also a goodstimulator of the T cell response in a majority of tuberculosispatients, it could be used either individually or as part of a“cocktail” of antigens in the design of a subunit vaccine againsttuberculosis.

The references cited herein are listed on the following pages and areexpressly incorporated by reference.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

-   1. Altschul, S. F., W. Gish, W. Miller, E. M. Myers, and D. J.    Lipman, 1990. Basic local alignment search tool. Journal of    Molecular Biology. 215:403–410.-   2. Andersen, A. B., and P. Brennan, 1994. Proteins and antigens of    Mycobacterium tuberculosis, p. 307–332. In B. R. Bloom (ed.),    Tuberculosis: Pathogenesis, Protection, and Control. ASM,    Washington, D.C.-   3. Andersen, P. 1994. Effective vaccination of mice against    Mycobacterium tuberculosis infection with a soluble mixture of    secreted mycobacterial proteins. Infect. Immun. 62:2536–2544.-   4. Berthet, F. X., J. Rauzier, E. M. Lim, W. Philipp, B. Gicquel,    and D. Portnoï, 1995. Characterization of the M. tuberculosis erp    gene encoding a potential cell surface protein with repetitive    structures. Microbiology, 141:2123–2130.-   5. Braibant, M., L. D. Wit, P. Peirs, M. Kalai, J. Ooms, A.    Drowart, K. Huygen, and J. Content, 1994. Structure of the    Mycobacterium tuberculosis antigen 88, a protein related to the    Escherichia coli PstA periplasmic phosphate permease subunit,    Infection and Immunity, 62:849–854.-   6. Fox, B. G., J. Shanklin, J. Ali, T. M. Loerh, and J.    Sanders-Loerb, 1994. Resonance Raman evidence for an Fe—O—Fe center    in stearoyl-ACP desaturase. Primary sequence identity with other    diiron-oxo proteins. Biochemistry 33:12776–12786.-   7. Fulco, A. J., and K. Bloch, 1962. Cofactor requirements for fatty    acid desaturation in Mycobacterium phlei. Biochim. Biophys. Acta.    63:545–546.-   8. Fulco, A. J., and K. Bloch, 1964. Cofactor requirements for the    formation of Δ9 unsatured fatty acids in Mycobacterium phlei. The    Journal of Biological Chemistry. 239:993–997.-   9. Haslov, K., A. Andersen, S. Nagai, A. Gottschau, T. Sorensen,    and P. Andersen, 1995. Guinea pig cellular immune responses to    proteins secreted by Mycobacterium tuberculosis. Infection and    Immunity, 63:804–810.-   10. Hatfull, G. F. 1993. Genetic transformation of mycobacteria.    Trends in microbiology, 1:310–314.-   11. Hermans, P. W. M., F. Abebe, V. I. O. Kuteyi, A. H. J.    Kolk, J. E. R. Thole, and M. Harboe, 1995. Molecular and    immunological characterization of the highly conserved antigen 84    from Mycobacterium tuberculosis and Mycobacterium leprae. Infection    and Immunity, 63:954–960.-   12. Izard, J. W., and D. A. Kendall, 1994. Signal peptides:    exquisitely designed transport promoters, Molecular Microbiology,    13:765–773.-   13. Jacobs, W. R., G. V. Kalpana, J. D. Cirillo, L.    Pascopella, S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta,    and B. R. Bloom, 1991. Genetic systems for mycobacteria. Methods    enzymol. 204:537–555.-   14. Kashiwabara, Y., H. Nakagawa, G. Matsuki, and R. Sato, 1975.    Effect of metal ions in the culture medium on the stearoyl-Coenzyme    A desaturase activity of Mycobacterium phlei. J. Biochem.    78:803–810.-   15. Kashiwabara, Y., and R. Sato, 1973. Electron transfer mechanism    involved in stearoyl-coenzyme A desaturation by particulate fraction    of Mycobacterium phlei. J. Biochem. 74:405–413.-   16. Keegstra, K., and L. J. Olsen, 1989. Chloroplastic precursors    and their transport across the envelope membranes. Ann. Rev. Plant    Physiol. Plant Mol. Biol. 40:471–501.-   17. Laemmli, U. K. 1970. Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature (London).    227:680–685.-   18. Lee, B. Y., S. A. Hefta, and P. J. Brennan, 1992.    Characterization of the major membrane protein of virulent    Mycobacterium tuberculosis. Infection and Immunity. 60:2066–2074.-   19. Legrand, P., and A. Bensadoun, 1991. Stearyl-CoA desaturase    activity in cultured rat hepatocytes. Biochimica et Biophysica Acta.    1086:89–94.-   20. Lim, E. M., J. Rauzier, J. Timm, G. Torrea, A. Murray, B.    Gicquel, and D. Portnoï, 1995. Identification of Mycobacterium    tuberculosis DNA sequences encoding exported proteins by using phoA    gene fusions. Journal of Bacteriology. 177:59–65.-   21. Pal, P. G., and M. A. Horwitz, 1992. Immunization with    extracellular proteins of Mycobacterium tuberculosis induces    cell-mediated immune responses and substential protective immunity    in a guinea pig model of pulmonary tuberculosis. Infection and    Immunity. 60:4781–4792.-   22. Romain, F., A. Laqueyrerie, P. Militzer, P. Pescher, P.    Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G.    Marchal, 1993. Identification of a Mycobacterium bovis BCG 45/47    —kilodalton antigen complex, an immunodominant target for antibody    response after immunization with living bacteria. Infection and    immunity 61:742–750.-   23. Sakamoto, T., H. Wada, I. Nishida, M. Ohmori, and N.    Murata, 1994. Δ9 acyl lipid desaturases of cyanobacteria. J. Biol.    Chem. 269:25576–25580.-   24. Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989. Molecular    cloning-A laboratory manual. Cold Spring Harbor Laboratory Press.    Cold Spring Harbor, N.Y.-   25. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing    with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA,    74:5463–5467.-   26. Shanklin, J., and C. Somerville, 1991.    Stearoyl-acyl-carrier-protein desaturase from higher plants is    structurally unrelated to the animal and fungal homologs. Proceeding    of the National Academy of Science of the United States of America.    88:2510–2514.-   27. Shanklin, J., E. Whittle, and B. G. Fox, 1994. Eight histidine    residues are catalytically essential in a membrane-associated iron    enzyme, stearoyl-CoA desaturase, and are conserved in alkane    hydroxylase and xylene monooxygenase. Biochemistry. 33:12787–12794.-   28. Snapper, S. B., B. R. Bloom, and J. W. R. Jacobs, 1990.    Molecular genetic approaches to mycobacterial investigation, p.    199–218. In J. McFadden (ed.), Molecular Biology of the    Mycobacteria. Surrey University Press, London.-   29. Sorensen, A. L., S. Nagai, G. Houen, P. Andersen, and A. B.    Andersen, 1995. Purification and characterization of a    low-molecular-mass T-cell antigen secreted by Mycobacterium    tuberculosis. Infection and Immunity 63:1710–1717.-   30. Southern, E. M. 1975. Detection of specific sequences among DNA    fragments separated by gel electrophoresis. J. Mol. Biol.    98:503–517.-   31. Studier, W., A. H. Rosenberg, J. J. Dunn, and J. W.    Dubendorff 1990. Use of T7 RNA polymerase to direct expression of    cloned genes. Methods in Enzymology 185:60–89.-   32. Thole, J. E. R., and R. v. d. Zee 1990. The 65 kD antigen:    molecular studies. on a ubiquitous antigen, p. 37–66. In J. McFadden    (ed.). Molecular Biology of the mycobacteria. Surrey University    Press, London.-   33. Wheeler, P. R., and C. Ratledge. 1994. Metabolism of    Mycobacterium tuberculosis, p. 353–385. In B. R. Bloom (ed.).    Tuberculosis: Pathogenesis, Protection, and Control, ASM.    Washington, D.C.-   34. Young, D., T. Garbe, R. Lathigra and C. Abou-Zeid, 1990. Protein    antigens: structure, function and regulation, p. 1–35. In J.    McFadden (ed.), Molecular biology of mycobacteria. Surrey University    Press, London.-   35. Young, R. A., B. R. Bloom, C. M. Grossinsky, J. Ivany, D.    Thomas, and R. W. Davis, 1985. Dissection of the Mycobacterium    tuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci.    USA 82:2583–2587.

1. A purified nucleic acid that hybridizes with a purified DNA codingfor an enzyme from the class II diiron-oxo protein family and comprisingthe nucleic acid sequence of SEQ ID No. 1, under stringent conditionscomprising prehybridization and hybridization in RHB buffer and washinga 65° C. as follows: 2 washes with 2×SSPE, SDS 1% of 15 min each, onewash with I×SSPE, SDS 0.1% of 10 min, and two washes with 0.7×SSPE, SDS0.1% of 15 min each.
 2. The purified nucleic acid according to claim 1,having 8 to 40 nucleotides in length.